Arunasalam Navaraj

CARPs Are Ubiquitin Ligases That Promote MDM2-independent p53 and Phospho-p53ser20 Degradation

Caspase 8/10-associated RING proteins (CARPs) are a recently described family of protein ubiquitin ligases that interact with and negatively regulate death receptor-mediated apoptosis. Because CARPs are overexpressed in cancer and their silencing reduces cell viability and sensitizes tumor cells to chemotherapeutic agents, we investigated their relationship to p53 tumor suppressor signaling. p53 is a major determinant of chemosensitivity, and its levels are increased following DNA damage through N-terminal phosphorylation and inhibition of degradation. Although p53 is well known to be negatively regulated by several ubiquitin ligases including MDM2, none are known to target phosphorylated p53 for degradation. CARPs physically interact with and ubiquitinate p53, targeting it for degradation in the absence of MDM2. Serine 20-phosphorylated p53 is also ubiquitinated by CARPs. CARP silencing stimulates p53 expression and promotes downstream effects, including transcriptional activation and tumor suppression.

The p53 target Plk2 interacts with TSC proteins impacting mTOR signaling, tumor growth and chemosensitivity under hypoxic conditions

Tuberous sclerosis complex 1 (TSC1) inhibits mammalian target of rapamycin (mTOR), a central promotor of cell growth and proliferation. The protein product of the TSC1 gene, hamartin (referred to as TSC1) is known to interact with Polo-like kinase 1 (Plk1) in a cell cycle regulated, phosphorylation-dependent manner. We hypothesized that the p53 target gene, Plk2, is a tumor suppressor, mediating its tumor suppressor function through interactions with TSC1 that facilitate TSC1/2 restraint of mTOR under hypoxic stress. We found that human lung tumor cells deficient in Plk2 grew larger than control tumors, and that Plk2 interacts with endogenous TSC1 protein. Additionally, C-terminal Plk2-GST fusion protein bound both TSC1 and TSC2 proteins. TSC1 levels were elevated in response to Adriamycin and cells transiently over-expressing Plk2 demonstrated decreased phosphorylation of the downstream target of mTOR, ribosomal protein p70S6 kinase during hypoxia. Plk2 levels were inversely correlated with cytoplasmic p70S6K phosphorylation. Plk2 levels did not increase in response to DNA damage (Adriamycin, CPT-11) when HCT 116 and H460 cells were exposed to hypoxia. TSC1-deficient mouse embryonic fibroblasts with TSC1 added back demonstrated decreased S6K phosphorylation, which was further decreased when Plk2 was transiently over-expressed. Interestingly, under normoxia, Plk2 deficient tumor cells demonstrated increased apoptosis in response to various chemotherapeutic agents including CPT-11 but increased resistance to apoptotic death after CPT-11 treatment under hypoxia, and tumor xenografts comprised of these Plk2-deficient cells were resistant to CPT-11. Our results point to a novel Plk2-TSC1 interaction with effects on mTOR signaling during hypoxia, and tumor growth that may enable targeting Plk2 signaling in cancer therapy.

Repression of c-Myc and inhibition of G1 exit in cells conditionally overexpressing p300 that is not dependent on its histone acetyltransferase activity.

p300 and cAMP response element binding protein (CREB)-binding protein (CBP) are two highly homologous, conserved transcriptional coactivators, and histone acetyltransferases (HATs) that link chromatin remodeling with transcription. Cell transformation by viral oncogene products such as adenovirus E1A and SV40 large T antigen depends on their ability to inactivate p300 and CBP. To investigate the role of p300 in cell-cycle progression, we constructed stable rat cell lines, which conditionally overexpress p300 from a tetracycline-responsive promoter. When p300 was induced in these cells, serum-stimulated S-phase entry was significantly inhibited. The inhibition of S-phase induction was associated with down-regulation of c-Myc, but not of c-Fos or c-Jun. Simultaneous overexpression of c-Myc and p300 before serum stimulation reversed the inhibition of S-phase induction to a significant level, indicating that the inhibition of c-Myc to a large extent is responsible for the p300 inhibition of G1 exit. Similar studies with stable rat cell lines that overexpress a mutant p300, which lacks the HAT activity, showed that the intrinsic HAT activity of p300 is not required for the negative regulation of c-Myc or G1. These findings, and our previously published results (Kolli, S., Buchmann, A. M., Williams, J., Weitzman, S. & Thimmapaya, B. (2001) Proc. Natl. Acad. Sci. USA 98, 4646–4651), establish an important negative regulatory role for p300 in c-Myc expression that may be important in maintaining the cells in the G0/G1 phase of the cell cycle.

Replication stress, defective S-phase checkpoint and increased death in Plk2-deficient human cancer cells.

We previously reported that the Polo-like Kinase 2 gene (Plk2/Snk) is a direct target for transcriptional regulation by p53 and that silencing Plk2 sensitizes cancer cells to Taxol-induced apoptosis. Our goals have been to better understand why Plk2 is regulated by p53 and how Plk2 signals protection from cell death through checkpoint activation. We found that following knock-down of Plk2 in wild-type p53 expressing H460 human non-small cell lung cancer cells there was a significant increase in cell death observed in aphidicolin-treated cells and a further increase after release from aphidicolin-block. The highest levels of cell death were observed when Plk2-deficient cells were released from both aphidicolin and etoposide treatment. These results suggested that a defective S-phase checkpoint may contribute to enhanced sensitivity of Plk2-deficient cells to replication stress. Consistent with this hypothesis, we observed higher levels of Serine 139 H2AX phosphorylation in Plk2-deficient as compared to control cells before and after aphidicolin treatment indicating that there is more DNA damage when Plk2 is depleted. We also observed higher levels of Chk1 protein in Plk2-deficient cells that were associated with reduced levels of Serine 317-phosphorylated Chk1. In aphidicolin-treated cells, there were lower levels of Serine 317-phosphorylated Chk1 when Plk2 was knocked-down. Plk2 was demonstrated to interact with Chk2, Chk1, Serine 317-phoshorylated Chk1 and p53. Thus, increased cell death observed after aphidicolin treatment and release in Plk2-deficient cells may result from both higher levels of replication stress-induced DNA damage and a dysfunctional S-phase checkpoint.

Tumorigenic Conversion of Primary Human Esophageal Epithelial Cells Using Oncogene Combinations in the Absence of Exogenous Ras

To investigate pathways of human esophageal squamous cell transformation, we generated esophageal tumor cells using human telomerase- and SV40-immortalized primary esophageal epithelial cells (EPC2) by overexpression of selected combinations of oncogenes. H-Ras, c-Myc, or Akt, but not epidermal growth factor receptor (EGFR), induced transformed colonies in soft agar. By contrast, bioluminescence imaging of genetically altered immortalized esophageal cells revealed that Akt, EGFR, or H-Ras, but not c-Myc, resulted in tumor formation in immunodeficient mice. H-Ras-driven tumors showed highly tumorigenic phenotypes with 2.6 +/- 0.6 days for doubling, whereas Akt and EGFR tumors doubled every 9.5 +/- 1.6 and 6.1 +/- 1.2 days, respectively. H-Ras-driven tumors expressed the hypoxia-inducible factor target Glut1, whereas Akt- or EGFR-driven tumors had evidence of angiogenesis and no detectable Glut1 expression. Proliferation rates among these tumors were similar, but there was reduced apoptosis in the more aggressive H-Ras-driven tumors that also developed aneuploidy and multiple centrosomes. c-Myc overexpression did not result in tumorigenic conversion but introduction of Bcl-XL into c-Myc-expressing cells generated tumors. Although cytokeratin expression was typical of squamous carcinoma, gene expression profiling was done to compare the four different types of engineered tumors with human esophageal squamous cell carcinomas and adenocarcinomas. Interestingly, c-Myc plus Bcl-XL transformants mimicked squamous carcinomas, whereas H-Ras-, EGFR-, and Akt-driven tumors were similar to adenocarcinomas in their molecular profiles. These genetically engineered models may provide new platforms for understanding human esophagus cancer and may assist in the evaluation of new therapies.

Quinacrine sensitizes hepatocellular carcinoma cells to TRAIL and chemotherapeutic agents.

Quinacrine has been widely explored in treatment of malaria, giardiasis, and rheumatic diseases. We find that quinacrine stabilizes p53 and induces p53-dependent and independent cell death. Treatment by quinacrine alone at concentrations of 10–20 mM for 1–2 d cannot kill hepatocellular carcinoma cells, such as HepG2, Hep3B, Huh7, which are also resistant to TRAIL. However, quinacrine renders these cells sensitive to treatment by TRAIL. Co-treatment of these cells with quinacrine and TRAIL induces overwhelming cell death within 3–4 h. Levels of DR5, a pro-apoptotic death receptor of TRAIL, are increased upon treatment with quinacrine, while levels of Mcl-1, an anti-apoptotic member of the Bcl-2 family, are decreased. While the synergistic effect of quinacrine with TRAIL appears to be in part independent of p53, knockdown of p53 in HepG2 cells by siRNA results in more cell death after treatment by quinacrine and TRAIL. The mechanism by which quinacrine sensitizes hepatocellular carcinoma cells to TRAIL and chemotherapies, and the potential for clinical application currently are being further explored. Lastly, quinacrine synergizes with chemotherapeutics, such as adriamycin, 5-FU, etoposide, CPT11, sorafenib, and gemcitabine, in killing hepatocellular carcinoma cells in vitro and the drug enhances the activity of sorafenib to delay tumor growth in vivo.

Quinacrine synergizes with 5-fluorouracil and other therapies in colorectal cancer.

Although treatments have improved patient prognosis in surgically resectable colorectal cancer, new effective drugs with improved safety profiles are needed to improve the currently poor outcomes of patients with recurrent or metastatic colorectal cancer. Quinacrine, a small molecule anti-malarial agent that has activity in giardiasis, lupus, prion disease, and used as a means of non-surgical sterilization, has shown cytotoxic activity across a broad range of cancers. Here, we evaluate the potential of adding quinacrine to anticancer chemotherapeutics and targeted agents as a potential novel combinatorial therapy for advanced colon cancer. We show that quinacrine synergizes with 5-fluorouracil and significantly enhances the cytotoxicity of sorafenib in a panel of 10 human colorectal cancer cell lines, including those with KRAS mutations protein gel blot analysis confirmed that quinacrine’s anticancer activity partially arises from its ability to stabilize p53 and lower anti-apoptotic protein levels. In a series of in vivo studies, quinacrine monotherapy lowered the tumor load of nu/nu mice bearing human colorectal cancer xenografts. In combination, quinacrine and 5-Fluorouracil significantly delayed tumor growth of a variety of different xenografts, as compared to each agent administered alone. Our results suggest that the administration of quinacrine in combination with chemotherapeutic agents and targeted agents should be further explored in patients with recurrent, locally advanced, or metastatic colorectal cancer.

Identifying Circulating Tumor Stem Cells That Matter: The Key to Prognostication and Therapeutic Targeting

TO THEEDITOR: We read with interest the article by Iinuma et al 1 that describes their study examining the clinical significance of cancer stem cells in the peripheral blood of patients with colorectal cancer (CRC). The defining characteristics of a circulating cancer stem cell (CTSC) are its capacity for self-renewal and for initiation of distant metastases; some of these cells are also resistant to traditional chemo- therapy. 2,3 The concept that circulating tumor stem cells can be iden- tified and targeted is attractive and has major diagnostic, prognostic, and therapeutic implications for patients with metastatic cancer. However, the identification of authentic CTSCs continues to be a challenge because of the lack of a clear understanding of the biologic heterogeneity of tumor stem-cell populations and also because of the lack of characterization of surface markers that predict clinically im- portant subsets of CTSCs, such as those with aggressive malignant potential or drug resistance.

Relationship between E1A binding to cellular proteins, c-myc activation and S-phase induction

We recently showed that p300/CREB-binding protein (CBP) plays an important role in maintaining cells in G0/G1 phase by keeping c-myc in a repressed state. Consistent with this, adenovirus E1A oncoprotein induces c-myc in a p300-dependent manner, and the c-myc induction is linked to S-phase induction. The induction of S phase by E1A is dependent on its binding to and inactivating several host proteins including p300/CBP. To determine whether there is a correlation between the host proteins binding to the N-terminal region of E1A, activation of c-myc and induction of S phase, we assayed the c-myc and S-phase induction in quiescent human cells by infecting them with Ad N-terminal E1A mutants with mutations that specifically affect binding to different chromatin-associated proteins including pRb, p300, p400 and p300/CBP-associated factor (PCAF). We show that the mutants that failed to bind to p300 or pRb were severely defective for c-myc and S-phase induction. The induction of c-myc and S phase was only moderately affected when E1A failed to bind to p400. Furthermore, analysis of the E1A mutants that fail to bind to p300, and both p300 and PCAF suggests that PCAF may also play a role in c-myc repression, and that the two chromatin-associated proteins may repress c-myc independently. In summary, these results suggest that c-myc deregulation by E1A through its interaction with these chromatin-associated proteins is an important step in the E1A-mediated cell cycle deregulation and possibly in cell transformation.

Cooperation between BRCA1 and p53 in repair of cyclobutane pyrimidine dimers

DNA repair defects can predispose to cancer development and progression. We previously showed that the breast and ovarian cancer susceptibility gene product BRCA1, through p53, upregulates expression of the XPE gene DDB2 encoding the nucleotide excision repair protein p48. Both XPE and XPC are p53 target genes containing p53 response elements. To further explore the role of BRCA1 and p53 in repair of photoproducts, we eliminated wild-type p53 from U2OS osteosarcoma cells and found that cyclobutane pyrimidine dimer (CPD) repair was markedly impaired following UV damage whereas repair of 6-4 photoproduct (6-4 PP) occurred efficiently. Overexpression of p53 in p53-null Calu-6 cells also enhanced CPD repair. In HCC1937 breast cancer cells, harboring mutant BRCA1 and p53 genes, repair of CPD was markedly impaired. Reintroduction of either p53 or BRCA1 using adenovirus vectors into HCC1937 alone had little effect on repair of CPD whereas the combination of p53 and BRCA1 resulted in efficient repair of CPD. Thus there appears to be a cooperative effect between p53 and BRCA1 that may involve induction of repair proteins, inhibition of p53-induced cell death by BRCA1 with altered p53 selectivity towards repair pathways and/or p53-independent effects of BRCA1 on CPD repair.